CN102516393A - Insulin mimetic peptide fusion protein, mutant and applications thereof - Google Patents

Insulin mimetic peptide fusion protein, mutant and applications thereof Download PDF

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CN102516393A
CN102516393A CN2011103997259A CN201110399725A CN102516393A CN 102516393 A CN102516393 A CN 102516393A CN 2011103997259 A CN2011103997259 A CN 2011103997259A CN 201110399725 A CN201110399725 A CN 201110399725A CN 102516393 A CN102516393 A CN 102516393A
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CN102516393B (en
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张海涛
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Xuanzhu Biopharmaceutical Co Ltd
Beijing Xuanzhu Kangming Biological Pharmaceutical Co Ltd
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Abstract

The invention relates to an amino acid sequence of a fusion protein for treating human type I and type II diabetes, and a production method and an application thereof. The invention relates to a noninsulin diabetes treatment method which can be used for avoiding insulin resistance. The fusion protein related to the invention is formed by fusing an insulin mimetic peptide and an IgG-Fc or IgG-Fc mutant through a connecting peptide, so that the in-vivo half-life of the insulin mimetic peptide can be prolonged remarkably while the blood sugar lowering activity of the insulin mimetic peptide is kept. The mutant of IgG-Fc can be used for further prolonging the in-vivo half-life of the fusion protein.

Description

Insulin-simulated peptide fusion protein and two mutants and application thereof
Invention field
The present invention relates to the prevention and the medicine of people's mellitus.Concrete, the present invention provides a kind of fusion rotein that is used to detect and treat people I type and type ii diabetes.Said fusion rotein contains insulin-simulated peptide (IMP) and human IgG-Fc (hinge area-CH2-CH3) albumen and/or IgG-Fc two mutants (T250Q/M428L; QL and T307Q/N434A; QA), can effectively reduce diabetic subject's glucose level, increase the two mutants of insulin-simulated peptide intravital transformation period, especially IgG-Fc (QL/QA), can further increase the internal metabolism transformation period of insulin-simulated peptide, reduce the injection frequency of medicine.
Background technology
The Along with people's growth in the living standard, the increase of aging population and fat incidence, the sickness rate of mellitus is ascendant trend year by year.Mellitus are to cause human dead main diseases therefore, nearly 200,000,000 patients in the whole world.Mellitus reach 2% at the sickness rate of China, and according to statistics, at present existing 3,980 ten thousand diabetics of China occupy the whole world second.Expect 2025, Chinese diabetic subject will reach 5,900 ten thousand people.China has more than 100 ten thousand newly-increased cases at present every year approximately.Wherein the type i diabetes patient accounts for 10%, and the type ii diabetes patient accounts for 90%.Estimate according to the expert, about 7,000,000,000 yuans of the market capacity of present domestic diabetes medicament, the data analysis of announcing according to the Ministry of Health shows that the coming years, the Absatzvolumen of China's mellitus class medicine will be above 10,000,000,000 yuans.
Mellitus mainly be divided into type i diabetes and type ii diabetes.Wherein, the complicated T cell that forms when being a kind of children's of being everlasting or teenager of type i diabetes relies on autoimmune disorder (Juneia and Palmer, Autoimmunity.1999; 29 (1): 65-83).Be that autoimmune destruction by beta Cell of islet causes and follows corresponding insulin deficit and to the dependence of exogenous insulin.The remarkable minimizing that monocyte is invaded the quality and quantity of profit and functional β cell to the focus property of pancreatic endocrine cell and tissue is the histopathologic characteristics of this disease of diagnosis, but the degree interindividual variation of these damages is remarkable.Autoimmunity destroys and to make the T cell invade to moisten to pancreas islet (islets of Langerhans), and apoptosis (people such as Lee, the Mol Genet Metab.2004 of β cell appears in the result; 83 (1-2): 82-92; Mandrup-poulsen, Biochem Pharmacol.2003; 66 (8): 1433-1440; Scsti, Ann Med.2002; 34 (6) 444-450; People such as Mathis, Nature.2001; 414 (6865) 792-798).For research mellitus potential molecular mechanism, set up several animal models.For example, with U-9889 (streptozotocin) (STZ) the cell-mediated inflammation of rat or mouse simulation visible T-in diabetic patient of inductive insulin deficit property and the destruction of islet cells.In addition; The mouse of non-obese diabetes (NOD) is the model of another research autoimmune disorder; Pancreas islet-antigenic activity T cellular infiltration pancreas islet and kill pancreatic wherein; And/or cause death (Aanderson and Bluestone, the Annu Rev Immunol.2005 that inflammatory process causes beta Cell of islet; 23:447-485).
Insulin treatment is the main means of intervening type i diabetes.Through activating insulin receptor, glucose level in the control agent.The acceptor of Regular Insulin is a tetramer, is connected through disulfide linkage with two β subunits by two α subunits.Two α subunits are positioned at the outside of cytoplasmic membrane, and the binding site of Regular Insulin is arranged on it; Two β subunits are transmembrane proteins, play Role in Plant Signal Transduction.When no Regular Insulin combined, the LCK of acceptor did not have activity.After Regular Insulin combined with the α subunit of acceptor and changed the configuration of β subunit, LCK just was activated, but two reactions of catalysis after the activation:
1, make the tyrosine residues phosphorylation of β subunit specific site in the tetramer mixture, this process is called autophosphorylation (autophosphorylation);
2, (insulin receptor substrate has tens tyrosine residues phosphorylations of vital role on IRSs), and the IRSs of phosphorylation can combine and activate the downstream effect thing and come blood sugar regulation with IRS.
Type ii diabetes is a kind of usually at the polygene malfunction of adulthood diagnosis, and it is characteristics with three main abnormal that cause hyperglycemia: 1. peripheral insulin resistance, 2. excessive generation, the 3. pancreas beta cell dysfunction of liver starch.Insulin resistant is meant that peripheral tissues mainly is the reaction reduction of Skeletal Muscle Cell to Regular Insulin, causes glucose transport to arrive obstacle (Kahn and Goldfine, the Jdiabetes Complications.1993 of these tissues; 7 (2): 92-105; People such as Weyer, Jclin Invest.1999; 104 (6): 787-794).Insulin resistant can occur in liver equally, causes in liver Regular Insulin can not suppress generation (Kahn and Goldfine, the Jdiabetes Compliations.1993 of liver glucose effectively; 7 (2): 92-105; People such as Lam, Am.Jphysiol Endocrinol Metab.2002; 283 (4) E682-E691).The result of insulin resistant is the increase in demand of body to Regular Insulin.At the commitment of insulin resistant, through increasing the compensatory mechanism that insulin secretion causes that β cell quality increases in the pancreas.These patients' glucose level still can maintain (Bonner Weir, Trends Endocrinol Metab.2000 in the normal range; 11 (9): 375-378; Bonner-Weir, Endocrinology.2000; 141 (6): 1926-1929).Many researchs show that if can keep the compensation of pancreas beta cell, insulin resistant itself also is not enough to excite outbreak (people such as Weyer, Diabetes.1999,48 (11): 2197-2203) of mellitus.But insulin resistant will not be corrected and become for a long time seriously, causes beta cell depleted to the excess demand of Regular Insulin, and insulin secretion reduces, and hyperglycemia symptom and significantly mellitus (DeFronzo, Diabetes.1988 occur on an empty stomach; 37 (6): 667-687; People such as Kahn, J Nutr.2001; 131 (2): 354S; People such as Weyer, J Clin Invest.1999; 104 (6): 787-794).
The conventional treatment of type ii diabetes comprises goes on a diet and takes exercise and use sulfo group urea (sulphonylureas), metformin and Regular Insulin to carry out the intervention of medicine property.But these treatments usually all can't stop the long-term decline of glycemic control and the dysfunction of β cell (people such as Matthews, Diabet Med.1998 in Most patients; 15 (4): 297-303; People such as Turner, JAMA.1999; 281 (21): 2005-2012) finally also can't keep blood glucose balance to the stepped treat-ment that type ii diabetes adopted clinically; For Most patients; From going on a diet and tempering single factor pharmacological agent, become inevitable process (people such as Turner, JAMA.1999 from the Zhongdao insulinize of combination therapy; 281 (21): 2005-2012; Gerich, Eur J Clin Invest.2002; 32Suppl 3:46-53).
Insulin receptor is crossed over the dimer protein that disulfide linkage that subunit forms links to each other by the film of striding of the outer subunit of the born of the same parents of two 135-kDa and two 95-kDa; Contain by upper reaches binding partner activated intracellular tyrosine kinase domain (Ebina; Y. wait people, Cell.1985; 40,747-758 and Ullrich, people such as A., Nature.1985; 313,756-761).After Regular Insulin is attached to acceptor; Activate a series of metabolism and mitosis reaction; These reactions be by comprise IRS1,2 and several extracellular protein matrix of Shc on Tyrosylprotein kinase generation phosphorylation and produce (White, M.F., Diabetologia.1997.40; Suppl.2, S2-S17).IRS1 and IRS2 are main IRSs, cause carbohydrate metabolism, and at different organs unique and eclipsed role are arranged.In addition, having very directly of the modification of IRS1 and IRS2 and mellitus concerns.(Yamauchi, people such as T., Mol.Cell Biol.1996; 16,3074-3084 and Withers, people such as D.J., Nature.1998; 391,900-904) .Thirone etc. has found the specific function of IRS1 and IRS2 in the preceding myocyte system (L6 cells) of mouse, and has carried out summing up (Thirone, people such as A.C., Trends Endocrinol.2006; Metab.17,72-78).
But IRS1 and IRS2 also do not come to a conclusion to the Relative Contribution of insulin signaling and insulin resistant development.Most of document of delivering in this field shows that IRS1 is main substrate, thus stimulated muscle and fatty tissue to the transportation of glucose, and in liver IRS1 and IRS2 in insulin signaling and metabolic complementary action.In contrast SHC, the insulin metabolism signal can not occur participating in directly, but in insulin-induced mitotic division, play keying action (Thirone, people such as A.C., Endocr.J.2000; 47,373-381).IRS and Shc start mitogenesis activation pathway kinases/extracellular regulated protein kinase (MAPK/ERK) approach, comprise that activating ERK1/2 causes genetic transcription and protein translation and cell growth.Other are PI 3 kinases (PI3-kinases) approach by IRS protein activated signal path; Comprise the activation of PKB; The necessary proteinic activation (White of metabolism that causes glycogen synthetase and other enzymes/acute Regular Insulin; M.F.Diabetologia.1997.40, Suppl.2, S2-S17).
Through display technique of bacteriophage obtained can the bound insulin acceptor synthetic peptide (20～40 of amino acid quantity) (Pillutla, people such as R.C., J.Biol.Chem.2002; 277,22590-22594).These polypeptide can be attached to three sites ( site 1,2 and 3) on the insulin receptor.The Regular Insulin binding site overlaid on 1 and 2 two site and the insulin receptor wherein, and combine polypeptide to be combined near the site 1 at the insulin receptor in site 3, form nearest homodimer.Wherein manyly can activate insulin receptor, have higher tiring and specificity, maybe be through machine-processed lowering blood glucose level (Schaffer, people such as L., the Proc.Natl.Acad.Sci.U.S.A.2003 of a kind of similar Regular Insulin and its receptors bind; 100,4435-4439).
These insulin-simulated polypeptide can activate insulin receptor as Regular Insulin, reduce the glucose level of mouse.Their structure is simpler, volume is littler, can effectively prevent the generation of insulin resistant.They are that insulin substitution is treated good drug candidate, perhaps have the potential purposes with the Regular Insulin curing diabetic with combined, and can be used as the design template of novel peptide type medicine.In correlative study work; Some investigators find one 31 amino acid whose insulin-simulated peptides (IMP) under study for action; Can be affine with the insulin receptor height, and, have more weak some genetic expressions of promotion and promoting mitosis effect with respect to Regular Insulin.Discover that further on the one hand, IMP has the bound insulin receptor affinity similar with Regular Insulin, but different with the action pathway of Regular Insulin, this shows that they can coexist on insulin receptor.In addition, find the significant difference that exists between these two part inductive gene expression patterns, IMP promotes fissional function to be starkly lower than Regular Insulin.This shows that IMP has the blood sugar decreasing effect identical with Regular Insulin, can avoid insulin resistant, has littler promotion cell mitogen effect, and is more safe.
Usually polypeptide drug serum half-life in vivo is all shorter.Therefore need frequent administration, high-frequency medicine is injected, and has had a strong impact on patient's quality of life, brings higher treatment cost simultaneously.Therefore, how the peptide class is bioactive is increased in the intravital transformation period simultaneously not influencing, and is the focus of numerous scientists' researchs.Form fusion rotein like the GLP-1 that describes among WO 02/46227 or the WO 05/000892 and rHSA or human IgG-Fc, can significantly strengthen its transformation period in vivo.The transformation period prolongs in the body of fusion rotein, possibly form owing to the discharge of its molecular change ambassador kidney reduces.Yet the in vitro study of fusion rotein demonstrates lower effect (people such as Kim, Diabetes.2003; 52 (3): 751-759).This makes people make great efforts to develop in vivo longer medicine of transformation period in stable effect and the body.Therefore, need develop to the potential molecular mechanism of I type and type ii diabetes for mellitus but not effective therapeutic strategy that should the mechanism consequence.The ideal invention is effectively to treat mellitus to insulin receptor, can reduce the injection frequency of medicine again, reduces the drug use cost, avoids insulin resistant.
IgG-Fc is one of fusion rotein of transformation period in most widely used prolongation peptide class, cytokine and the soluble receptors class body, has compared many remarkable advantages with other proteins.It can prolong intravital serum half-life on the one hand, can form dimer in addition, and the probability of raising and targeted integration is more deep for the research of IgG-Fc on the other hand, therefore uses more safe.In IgG; Site (Fig. 1) mediation between CH2 on the Fc and CH3 territory and the interaction of newborn acceptor FcRn; Wherein combine to make endocytosis Fc fusion rotein be recycled to (Raghavan et al. in the blood flow from endosome; 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al, 2000, Annu Rev Immunol 18:739-766).Said process combines can reduce the kidney filtration behind the macromole, produces the interior serum half-life of body in 1～3 week.Being combined in the monoclonal antibody transhipment of Fc and FcRn also plays a crucial role.The site of the last combination of Fc FcRn also is bacterioprotein A and G bonded site.Usually can be used as protein purification with these proteic combining closely, use the mode of albumin A or Protein G affinity chromatography antibody purification Fc.In addition; The FcRn acceptor also is responsible for and will be transferred in neonatal enteron aisle and the adult enteric epithelium chamber with its bonded Fc fusion rotein, increases the cell of antibody Fc and penetration power (Ghetie and Ward, the Annu.Rev.Immunol. of tissue; 2000,18:739-766; Yoshida et al., Immunity, 2004,20 (6): 769-783).
In the mutation research process of people Fc γ, to combining some important residues of FcRn to study, the result shows that some amino acid whose sudden changes can significantly increase serum half-life.Hinton etc. have been mutated into other 19 kinds of common amino acid with three residues seriatim in people Fc γ 1.They find that the double mutant of some point mutation has increased the FcRn binding affinity, and wherein two sudden changes in T250Q/M428L site can significantly improve the avidity effect of Fc and FcRn, and then obviously improve the transformation period in vivo.In addition, Henry B.Lowman etc. can significantly strengthen serum half-life in the body equally through the T307Q/N434A site of sudden change IgG-Fc.These experiments have all obtained confirmation through the drug disposition metabolism experiment of monkey.In addition; Can also increase the transcytosis (Transcytosis) of fusion rotein or antibody and the permeability of tissue based on the sudden change that increases FcRn avidity; Increase the degree of exposure of in-vivo tissue system to medicine; Can bring into play drug action (Hinton et al., 2004, J.Biol.Chem.279 (8): 6213-6216 to greatest extent; Henry B.Lowman et al., Cancer Research.2010,70 (8): 3269-3277; Y Zheng et al, Clinical Pharmacology&Therapeutics.2010,89 (2), 283-290).
Summary of the invention
The present invention relates to prevent and treat the fusion rotein and the working method of I type and type ii diabetes.Compsn of the present invention comprises new fusion rotein and two mutants thereof.This fusion rotein contains Insulin mimetic peptides (IMP) molecule or its analogue or the fragment that merges with IgG heavy chain constant (Fc) district.This IgG is the people source, can be selected from IgG1, IgG2, IgG3 or IgG4.Fusion rotein of the present invention is called as IMP/IgG-Fc or IMP/IgG-Fc (T250Q/M428L in this article; QL), perhaps IMP/IgG-Fc or IMP/IgG-Fc (T307Q/N434A; QA).
In other aspects of the present invention, the two mutants of this fusion rotein is IMP/IgG-Fc (T250Q/M428L), contains one or two sudden changes of T250Q/M428L; Also can be IMP/IgG-Fc (T307Q/N434A; QA), comprise one or two sudden change, the perhaps combination of said mutation.
Fusion rotein of the present invention and two mutants thereof are effective to the treatment of I type and type ii diabetes in the experimenter.Likewise, the invention provides and be used to treat experimenter's the I type and the method for type ii diabetes, wherein use described fusion rotein and two mutants thereof to the experimenter if desired.
The present invention also provides the use Mammals to express and the microbial culture system, prepares the novel method of fusion rotein IMP/IgG-Fc of the present invention and IMP/IgG-Fc (T250Q/M428L) and/or IMP/IgG-Fc (T307Q/N434A).In these two kinds of systems; Culturing cell is cloned fusion rotein and the sudden change build of effective interior transformation period of extension body that comprises IMP/IgG-Fc and IMP/IgG-Fc (T250Q/M428L) and/or IMP/IgG-Fc (T307Q/N434A) with preparation, such as but not limited to IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA).Enhancing is to the serum residence time in the combination of FcRn acceptor and the extension body.
Divalence IMP/IgG-Fc of the present invention or IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) fusion rotein have distinct feature:
1) increased body-internal-circulation transformation period T1/2, especially the IgG-Fc two mutants (QL) of IMP or (QA) more can further increase the transformation period in the body;
2) compare with Regular Insulin, have lower irritation cell splitting ability, safer, can avoid insulin resistant;
3) fusion rotein forms and two body structures are more stable, with IgG-Fc two mutants (QL) or (QA) merges the permeability that increases medicine transcytosis (Transcytosis) and tissue, the exposure that the increase medicine is organized in vivo simultaneously;
4) through biological engineering method production, help obtaining the higher albumen of biological activity.The easy single stage method that is used for mass preparation is purified.Can fusion rotein of the present invention be offered the experimenter on demand in a variety of forms.
One side of the present invention provides a kind of experimenter's of being used for controlling blood sugar equilibrated IMP fusion rotein.
One side of the present invention provides a kind of IMP fusion rotein that is used to control experimenter's blood sugar concentration.
One side of the present invention provides a kind of IMP fusion rotein, and it activates insulin receptor through being different from the mode of Regular Insulin, promotes the blood sugar metabolism.
According to an aspect of the present invention, fusion rotein of the present invention has been avoided experimenter's insulin resistant, strengthens glucose tolerance and reduces fasting blood glucose level.
One side of the present invention provides a kind of fusion rotein, and this fusion rotein contains IMP polypeptide or analogue or its two mutants or its fragment or IgG polypeptide.
One side of the present invention provides a kind of heterology fusion rotein, and this fusion rotein contains IMP polypeptide or its varient that merges with the IgG polypeptide, wherein said IgG.
According to an aspect of the present invention, said IgG is the people source.
According to an aspect of the present invention, said IgG Fc is human IgG1, IgG2 or IgG4.
According to an aspect of the present invention, said IgG is the human IgG2.
According to an aspect of the present invention, the said IgG-Fc heterozygote that is humanized IgG 1-Fc, IgG2-Fc or IgG4-Fc.
According to an aspect of the present invention, said IMP polypeptide is selected from the group of being made up of insulin-simulated peptide (SLEEEWAQIECEVYGRGCPSE SFYDWFERQL) and fragment thereof and verivate.
According to an aspect of the present invention, said IMP is SLEEEWAQIECEVYGRGCPSESFYDWFERQL.
According to an aspect of the present invention, said verivate contain about 70% to about 95% with IMP (the Sequence ID NO.1) polypeptide of sequence that sequence is identical.
According to an aspect of the present invention, said IgG fragment contains at least 5 amino acid to 250 amino acid nearly.
According to an aspect of the present invention, said IgG contains the fragment or the verivate of Fc part or the Fc part of IgG.
One aspect of the present invention provides a kind of aminoacid sequence of the said heterology fusion rotein of encoding.
One aspect of the present invention provide a kind of aminoacid sequence that contains said fusion rotein through codon optimized carrier.
One aspect of the present invention provides a kind of host cell of the carrier transfection of optimizing through codon preference through the aminoacid sequence that contains said heterology fusion rotein.
One side of the present invention provides a kind of medicinal compsns, and said compsn contains above-mentioned heterology fusion rotein or comprises the carrier of optimizing through codon preference with the aminoacid sequence of the heterology fusion rotein of pharmaceutically acceptable carrier load.
According to an aspect of the present invention, said medicinal compsns is used to treat I type and type ii diabetes.
According to an aspect of the present invention, said medicinal compsns can be used through the mode that is selected from the group of being made up of topical application, oral, aerosol mode, peritoneal injection, intravenous injection or intramuscular injection.
According to an aspect of the present invention, said medicinal compsns is used for curee's the I type and the treatment of type ii diabetes, and said compsn contains and comprises and the IMP polypeptide of IgG polypeptide fusion or the heterology fusion rotein of its verivate or active fragments.
One aspect of the present invention provides a kind of experimenter's of treatment the I type and/or the method for type ii diabetes, and said method comprises fusion rotein or the said compsn or the said carrier of administering therapeutic significant quantity.
One aspect of the present invention provides said heterology fusion rotein to be used for treating or preventing the purposes of the medicine of I type and/or type ii diabetes.
According to an aspect of the present invention, said IgG is selected from the group of being made up of human IgG1, IgG2, IgG3 and IgG4.
Other characteristics of the present invention and meliority can embody through following detailed description.But; It should be understood that; Only with the mode of example provide describe in detail with specific embodiment showing embodiment of the present invention, this be because of for those skilled in the art from described detailed description in essence of the present invention and scope multiple change and improvement be conspicuous.
Description of drawings
From following detailed description, will be further understood that the present invention with reference to accompanying drawing, wherein:
Fig. 1: the expression plasmid that makes up coding IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA).1A shows the utilization round pcr, and the sequence with IMP and IgG-Fc increases from template separately respectively, is template with above-mentioned PCR product next, amplifies the IMP/IgG-Fc sequence with primer 1 and 4.Then, the cDNA with coding IMP/IgG-Fc fusion rotein is connected between the BmHI and EcoRV site of expression vector pcDNA3.1.Utilize the nucleotide site directed mutation method, produce the cDNA of coding IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) fusion rotein, use method same as described above that IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) are cloned into the pcDNA3.1 expression vector.The diagram that shows the IMP/IgG-Fc fusion rotein of forming by active IMP molecule and the IgG-Fc that comprises human IgG 4 CH (hinge, CH2 and CH3 part) among the 1B.All with the form secretion of homodimer, form space structure as shown in the figure during these protein expressions.
Fig. 2: demonstration be IMP/IgG-Fc, IMP/IgG-Fc (QL) or expression and the detection of IMP/IgG-Fc (QA) fusion rotein in the CHO-S cell.Merge transfection CHO-S cell with coding IgG-Fc, IMP/IgG-Fc, IMP/IgG-Fc (QL) or the proteic plasmid vector of IMP/IgG-Fc (QA), draw supernatant behind the transfection 48h, coagulate a strand purified fusion protein with A albumen agarose.The SDS-PAGE of (reduced) and non-reduced (Non-reduced) of reducing respectively analyzes, and dyes with Xylene Brilliant Cyanine G (Comasie Blue).Then, SDS-PAGE glue is transferred on the CAM, carried out Western blot and analyze.Above-mentioned film is surveyed with anti-mouse antibodies (1: 5000), and develops with ECL.The result shows that IMP/IgG-Fc, IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) are about the homodimer of 70KDa, and is consistent with theoretical molecular.Wherein, swimming lane 1,2,3 is respectively the SDS-PAGE of IMP/IgG-Fc, IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) reduced state.Swimming lane 5,6,7 is the SDS-PAGE of the non-reduced state of IMP/IgG-Fc, IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) fusion rotein.
Fig. 3: demonstration be that the RT-PCR that in the CHO-S cell, expresses of IMP fusion rotein detects.With growth conditions CHO-S cell implantation preferably 150mm 2Cultivate the expression plasmid DNA transfection that builds in inferior daily 80 μ g in the petridish.Dress dyes the back and screens with the G418 microbiotic, and behind transfection 48h, uses the total RNA extraction reagent box to separate total RNA.And use the cDNA that obtains through reverse transcription to carry out RT-PCR and analyze as template.What Fig. 3 showed is the RT-PCR product on 1% sepharose with the Goldenview colour developing.Wherein, swimming lane 1～6 is followed successively by contrast, DL2000Maker, blank, IMP-Fc, IMP-Fc (QA), IgG-Fc.
Fig. 4: demonstration be IMP/IgG-Fc, IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) fusion rotein are to the effect of the db/db model mouse of suffering from type ii diabetes.The db/db mouse in 4 weeks and/or 6 ages in week is through intramuscular injection IMP/IgG-Fc or IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) fusion rotein.Collect before the injection and serum 2,12,16 weeks of injection back.Active IMP level detects (data not shown) through IMP Elisa test kit.Inject the fasting blood glucose level (n=5～6, p＜0.001) that detected two groups of mouse in back 2 days first.
Fig. 5: demonstration be expression in vivo IMP/IgG-Fc, IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) fusion rotein are to the effect in the type i diabetes model of U-9889 inductive Regular Insulin defective.To the CD1 model mouse, every mouse intramuscular injection IMP/IgG-Fc, IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) fusion rotein 1nM.Preceding 2 days of injection fusion rotein is tried mouse and is received to strengthen injection STZ (55mg/kg, peritoneal injection), and accepts injection continuous 5 day every day the same day.Injection STZ after a couple of days, the contrast of injection tried mouse blood sugar increase significantly (>=17mM), but IMP/IgG-Fc, IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) handle is tried the occurrence frequency that low obvious mellitus were protected and shown to mouse.
Embodiment
The invention provides the fusion rotein and the method for prevention and treatment mellitus.This fusion rotein helps the blood sugar regulation level through insulin receptor.When individuality was used the fusion rotein of significant quantity, fusion rotein of the present invention was through activating insulin receptor and coherent signal path, the generation of treatment and prevent diabetes.
In one embodiment, the compositions and methods of the invention have prolonged the circulating half-life of IMP, have strengthened its effect.This is through providing the fusion rotein (IMP/IgG-Fc) that contains active IMP and IgG CH to realize.Said IMP peptide is naturally self to have embodiment.IgG is the people source.Humanized IgG can be selected from IgG1, IgG2 and IgG 4The IMP polypeptide can be the fragment of sequence own or verivate.The IMP polypeptide can be a SLEEEWAQIECEVYGRGCPSESFYDWFE RQL aminoacid sequence.
In another embodiment, fusion rotein of the present invention contains fragment or the IgG-Fc (QL) and IgG-Fc (QA) the two mutants fragment of IMP polypeptide, IMP fragment or verivate, or derivatives thereof.
But the present invention also provides the carrier of the excretory fusion rotein of the present invention of encoding, and said fusion rotein is used for the IMP polypeptide that Mammals is expressed divalence including, but not limited to active IMP and human IgG1/IgG2/IgG4-Fc aminoacid sequence; And active IMP-IgG Fc (QL) and IgG-Fc (QA) aminoacid sequence.Those skilled in the art can easily prepare any required IMP sequence in as the carrier of in this paper embodiment, describing or with identical method.Body outer cell line research combines I type and the interior intramuscular injection fusion rotein of type ii diabetes model mouse body to treat, and proves the human IMP fusion rotein IMP/IgG-Fc of reorganization, biological characteristics and the validity of IMP/IgG-Fc (QL) and IMP-IgG-Fc (QA).This treat-ment proves in serious I type and type ii diabetes model effectively.Effective in human and macrofauna through the experiment proof, and prove that through the intramuscular injection fusion rotein efficient is lower in rodent.In a word, the present invention is for preventing and treat the I type and type ii diabetes provides a kind of new method with protein therapeutic and gene therapy in mammals.
Fusion rotein of the present invention can be the IMP fragment, and itself and polypeptide have at least 60% or more identical sequence.Said sequence can have at least 70%, 80%, 90%, 95% or more identical sequence with the IMP polypeptide of form known; These form known comprise analogue, its verivate and its fragment.For example the sequence of these forms is open in USP 20070775642.The present invention also comprises the purposes that above-mentioned compsn is used for preventing and treating the medicine of mellitus, for example I type and type ii diabetes.The present invention also comprises a kind of medicinal compsns that is used for above-mentioned all purposes, for example preventative compsn.
The structure of fusion rotein is to merge IMP and a kind of new molecule of IgG-Fc molecular composition, and this molecular energy promotes the effect of IMP and be combined with the advantage of IgG-Fc that the interior transformation period of body that promptly increases IMP promotes fissional risk with reducing, and avoids insulin resistant.The invention provides the fusion rotein that has combined the IgG-Fc two mutants, said fusion rotein, for example fusion rotein combines IMP and IgG-Fc molecule to form the recruit, and this recruit has the similar effect of IMP and possesses the advantage of IgG-Fc molecule.Equally, the recruit that IMP and IgG Fc (QL) or IgG-Fc (QA) form, it has the advantage of described promotion IMP effect and IgG-Fc (QL) or IgG-Fc (QA) molecule, and the avidity of enhancing Fc and FcRn further increases the intravital transformation period.
Scope of the present invention comprises the variation that produces its chemical equivalence thing or chemically similar aminoacid sequence and do.The IgG-Fc fusion polypeptide that has an identical sequence with the IMP sequence and is confirmed to be applicable in the method for the present invention through test within the scope of the invention.USP (application number 20070775642, complete being incorporated herein by reference) has been described many insulin-simulated peptides.But the multiple polypeptides Lock-in varient among the present invention also can pass through sudden change, maybe can pass through synthetic, for example through as the protein engineering of rite-directed mutagenesis, this technology is that amino acid replacement well known in the art is technological.Can be replaced as L-Ala by another hydrophobic residue as glycocoll such as a hydrophobic residue, and alanine residue can be by the stronger leucine of hydrophobicity, Xie Ansuan, Isoleucine replacement.Electronegative amino acid such as aspartic acid can be replaced by L-glutamic acid, and positively charged amino acid such as Methionin can be by another positively charged amino acids Arginine replacements.
So, present invention resides in the IgG-Fc fusion polypeptide of change with a grain of salt on the aminoacid sequence or replacement.One or more amino acid are inserted in replacement with a grain of salt, and its reservation is replaced the identical chemical property of amino acid.The present invention includes the alternative sequence of conservative type, through not destroying the activity of original sequence after the conservative property replacement.The present invention expects that the IgG-Fc fusion polypeptide contains 1 or a plurality of D-amino acid.Expection N-end has the acetylizad polypeptide of one or more amino acid.It will be appreciated by those skilled in the art that and to make up polypeptide analog with multiple technologies; This analogue has required and the identical or similar character of corresponding polypeptide compound of the present invention, but in solubleness, stability and/or aspect the susceptibility of hydrolysis and enzymolysis, have better activity.For example, described in Morgan and the Gainor (Ann.Rep.Med.Chem., 24:243-252,1989).The embodiment of polypeptide analog is at United States Patent(USP) No. 5,643, describes in 873.Other are described the patent how to prepare and to use polypeptide analog and comprise (US5 for example, 786,322,5,753,226,5,654; 276,5,643,873,5,683,983,5; 677,280,5/767,075,5,668,110,5; 763,571,5,672,584, all above-mentioned documents are this complete being incorporated herein by reference).Also can be according to other technology preparations known in the art polypeptide analog of the present invention.For example, through use converts hydrogen group chemically for as the another kind of group of hydroxyl or the amino factor that changes side group handle IMP-Fc fusion polypeptide of the present invention.Analogue preferably includes to be the sequence of amino acid whose composition fully or to comprise amino acid and the hybrid of the amino acid of modification or other organic molecules.The present invention comprises hybrid and IMP-Fc fusion polypeptide equally, and for example, one of them nucleotide sequence combines with another sequence.
The present invention also comprises the identical active segment of having of IMP-Fc fusion polypeptide of the present invention, also comprises the segment that can be used as check polypeptide character and active research tool of fusion polypeptide.This peptide species preferably is made up of at least 5 amino acid.In preferred embodiments, they can be made up of 6～10,11～15,16～25,26～50,51～75,76～100 or 101～250 amino acid.Segment can include the sequence of one or more aminoacid deletion, for example, and the N-terminal amino acid in the compound sequence.
The present invention includes the fusion rotein of band sudden change, these amino acid mutations can occur in fusion rotein non-activity district part, thereby these amino acid mutations also can occur in the activity that the active zone part increases or reduced fusion rotein.In the present invention; Described in USP WO.05/00092 (this complete being incorporated herein by reference), the IgG-Fc part of technique known modification fusion rotein is to change (increase or reduce) immunogenicity (immunogenicity) or effector functions (effector function) by one of skill in the art.
Fusion rotein of the present invention can effectively use separately, but also can in pharmaceutical composition, use with other compositions like pharmaceutical carrier.Can combine fusion rotein of the present invention, can the soluble form above a type be used for the treatment target that receives like the mankind or animal, with prevention or treatment mellitus.
Fusion rotein of the present invention can pass through number of ways as medicinal application in the mankind or animal, is not limited to topical application, as inculcate in oral, spraying, the tracheae, peritoneal injection, intravenous injection, intramuscular injection and gene therapy.Application dosage depends on effect and the route of administration that needs of patients, expectation obtain.For example human amount of application can be 2nmol/kg body weight or 0.02～100nmol/kg body weight.When using gene therapy, people's DNA injection concentration can be 1 μ g/kg body weight or 0.1～100 μ g/kg body weight.Can be with body lipid body or viral transport vehicle with the fusion rotein transfered cell.Be applicable to that multiple transport vehicle of the present invention is known to those skilled in the art.Compsn of the present invention can every day, weekly or follow the doctor's advice and use the time that needs of continuing.
Medicinal compsns of the present invention can be used the method preparation to pharmaceutically acceptable carrier of patient with known preparation, thereby makes the Nucleotide of significant quantity or peptide molecule combine to form mixture with pharmaceutically useful carrier.The carrier that these are fit to, for example Lei Mingdun pharmacopedics (Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. is described in USA).On this basis; Said medicinal compsns can comprise combine with one or more pharmaceutically useful carriers or diluent and be present in the suitable pH value of tool and with the isoosmotic damping fluid of physiological liquid in active compound or material, for example composite nucleic acid molecule, peptide molecule or merge egg.Is known in conjunction with bioactive molecule and carrier or with both and thinner bonded method to those skilled in the art.Said compsn can comprise that being used to transport active compound arrives the targeting preparation (targeting agent) of organizing interior privileged site.
Compsn of the present invention can be treated as using ofhypoglycemic medicine or bound insulin with the treatment measure of known I type and/or type ii diabetes jointly.For example, the medicine of treatment mellitus can comprise: Victoza, Actos, Amaryl, avandia, DiaBeta, Diabinese, Dymelor, Glucophage, GlucophageXR, Glucotrol, GlucotrolXL, Glucovance, Glynase, PresTab, Glyset, Micronase, Orinase, Pandin, Precose, Starlix and Tolinase.For example, the Regular Insulin that is fit to comprises: Aspart, Insulin, Glargine (Lantus), Lispro (Humalog), NPH and Ultralente etc.
The present invention is applicable to the individuality of any this type of treatment of needs.These individualities might develop into mellitus or be diagnosed as the patient of mellitus recently or be diagnosed as the patient of mellitus.The present invention and prevention and to treat I type and type ii diabetes as described herein relevant.For example, such individuality can or have mellitus heredity history but the people of morbidity or be diagnosed as or be diagnosed as the people of mellitus recently not as yet for the people of obesity.The World Health Organization (WHO) is fat according to body-mass index (BMI) definition.BMI is with Kg body weight square getting divided by height rice number.Be that 18.5～25 body weight are normal.It is overweight that individual BMI is 25～30, equals or exceeds 30 for fat.These individualities also can be the people of the blood sugar normal value that is higher than same age and body weight (euglycemia can according to medical science bibliography conventional determining), although also be unlikely to be diagnosed as mellitus.But these individualities also can be mellitus heredity history to be arranged the people of also not developing into mellitus., blood glucose value can be diagnosed as mellitus when being higher than universally recognized normal range.
The present invention also provides the plasmid of new encoding fusion protein, and this fusion rotein coding contains through using people IMP and the human IgG-Fc (Fig. 1) that overlapping PCR (polymerase chain reaction) obtains.Legend demonstration IgG-Fc contains the constant heavy chain of IgG4 (comprising hinge, CH2 and CH3) in the zone.
The present invention also provides can produce leader and import and has been secreted into the method for going in the nutrient solution environment of extracellular after carrier can be expressed fusion rotein.As scheme to show that one section IgK secretion leading peptide sequence merges with the IMP sequence and guides the synthetic polypeptide to be secreted in the nutrient solution.In an embodiment of the present invention, one section rat immune globulin K (MUS IGK) leading peptide sequence (MVSTPEFLVFL LFWIPASRG) is secreted in the nutrient solution with IMP and IgG-Fc sequence fusion guiding synthetic polypeptide.Among each embodiment, this strategy has guaranteed in secretion process, after excision secretion leading peptide, will make the IMP of generation and the active histidine residues of IgG-Fc fusion rotein N end to merge.Representational IMP/IgG-Fc sees Fig. 2.This method can reach:
1) since the Fc fusion rotein with the dimeric form secretion of homogeneous, IMP-Fc fusion rotein long half time and, thereby its higher part affinity makes fusion rotein have higher tiring.
2) exist with dimeric precursor forms at cell surface owing to a large amount of insulin receptors, thereby strengthened the drug effect of IMP polypeptide.
3) simplified purge process.Utilize Protein A gel to accomplish in a step and purify, be beneficial to the separation and purification of fusion rotein.
This patent also discloses and has used mammalian expression system to prove the expression of the novel vector of above-mentioned fusion rotein.Be the ability of assessment vector expression and secretion IMP/IgG-Fc fusion rotein, with plamid vector transfection to the CHO-S cell.48h after the transfection extracts total RNA from the transfectional cell of expressing, with RT-PCR assessment Expression of Fusion Protein situation.Detect IMP-Fc fusion rotein and the expression of IgG-Fc reference protein on transcriptional level with gene-specific primer (Fig. 3).But not the transfection sample does not detect the expression of above-mentioned albumen on gene transcription level.
Chinese hamster ovary cell CHO-S cell lysate and the cell culture fluid of also having analyzed transfection with Western immunoblotting and anti-mouse antibody are to confirm the expression of fusion rotein on translation skill.Shown in Fig. 2 A, detect the Fc fusion rotein at nutrient solution and cell lysate.Can detect fusion rotein through RT-PCR reverse transcriptase polymerase chain reaction and Western immunoblotting.In conditioned medium and cell lysate, detect fusion rotein simultaneously and show fusion rotein synthetic justacrine in mammalian cell.CHO-S clone respectively transient transfection the IMP/IgG-Fc plasmid and the IMP-Fc-only plasmid of incremental change, 48h collecting cell nutrient solution after the transfection.From 50ml nutrient solution (inoculum density about 1.25 * 10 5Individual cell/ml, static cultivation in 2 days) utilizes Protein A gel single step purification fusion rotein (people such as Jungbauer, J Chromatogr.1989; 46:257-268) obtain about 300 μ g fusion roteins.Sample separates with non-reducing 2 kinds of gel electrophoresises and dyes through Coomassie brilliant blue through reduction respectively again and detected the band (Fig. 2) of an about 35KDa or about 70KDa respectively, shows that dimer IMP/IgG-Fc fusion rotein exists with native state.
In one embodiment, demonstration be IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) and effective in prevention and treatment I type and type ii diabetes are used for embodiment, 1. pre-diabetes db/db mouse is as the model of type ii diabetes; 2. the mouse that lacks Regular Insulin by U-9889 (STZ) inductive I type is as the type i diabetes model.The Db/db mouse lacks functional leptin receptor, therefore forms obesity, Hyperinsulinism (hyperinsulincmia) and when 4 to 6 ages in week, glucose occurs not tolerate, and tangible mellitus when 8 ages in week, occur.Therefore these mouse are widely used and are considered to the model of type ii diabetes.Through low dosage, repeatedly medication uses U-9889 (STZ) to induce the mouse that lacks Regular Insulin.U-9889 can destroy beta cell specifically, follows the cell-mediated infiltration of T-.Therefore these mouse are widely used and are considered to simulate the zootype of individual type i diabetes.Can obtain and can be applied to other mouse diabetes model of the present invention at present also has: the mouse model of high fat diet inductive insulin resistant; This mouse causes the body fat excess deposition because of excessively taking in fat, thereby forms obesity, the intolerance of tool insulin resistance and glucose.Another kind of animal model is non-obese diabetes (NOD) mouse.These mouse are good autoimmune type 2 diabetes mellitus (type i diabetes) models, wherein the pancreas islet antigenic activity T-cellular infiltration pancreas islet and kill islet cells, the process of causing inflammation makes islet cells dead (Anderson and Bluestone, 2005).
Use IMP/IgG-Fc through external injection for curing approach, IMP/IgG-Fc (QL) or IMP/IgG-Fc's (QA) is effective in prevention and treatment I type and type ii diabetes.It is effective that the for example IMP/IgG-Fc of any fusion rotein of the present invention that encodes, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) molecule are used for treatment.Through fusion rotein injection and to 4 age in week db/db tried the mouse injection and used above-mentioned fusion rotein.All is tried in the mouse, after injection for the first time, carry out the injection second time 1 week, and monitoring is tried the progress of mouse mellitus.The db/db mouse that heredity lacks leptin receptor is that (Leiter, FASEB are J.1989 for the type ii diabetes model of rodents; 3 (11): 2231-2241).As implied above, it is normal that the mouse of the age-matched of handling with IMP/IgG-Fc through fusion rotein treatment approach simultaneously (injection 1 week of back) when 16 ages in week demonstrates the blood sugar amount, and control group mice fasting plasma glucose (FBG) level has then exceeded normal range (Fig. 5).These results show that the treatment with IMP/IgG-Fc can prevent db/db mouse generation mellitus.Tried not have in the mouse generation of mellitus at the db/db that expresses IMP/IgG-Fc.
Through intramuscular injection, with IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) are posted to CD1 respectively and are tried in the mouse.The injection fusion rotein in, mouse has been carried out once follow-up injection, and begin to inject continuous 5 day every day STZ (55mg/kg, i.p.).The result finds; The blood sugar concentration of IgG-Fc control mice significantly rises; Blood sugar concentration has reached the level (17nM) of mellitus after several days, but IMP/IgG-Fc, and IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) mouse are protected and show the low incidence (Fig. 4) of obvious mellitus.
Medicine based on IgG-Fc has many advantages.Because fusion rotein can form the dimeric form of 70KDa, can't be removed at short notice by kidney, therefore have the longer in fact transformation period (Larrick and Fry, Hum Antibodies Hybridomas.1991; 2 (4): 172-189; People such as Weir, Biochem Soc Trans.2002; 30 (4): 512-516).Therefore, the big more natural IMP transformation period of IMP/IgG-Fc homodimer fusion rotein increases.The smaller polypeptides because some enzymes are degraded more easily (people such as Hupe-Sodmann, Regul Pept.1995; And the bigger IMP/IgG-Fc of molecular weight is not easy to be degraded 58 (3) .149-156).And the IMP/IgG-Fc dimer can increase the affinity of part, through preformed insulin receptor dimer and polymer (people such as George, Nat Rev Drug Discov.2002; (10): 808-820; People such as Dupuis, Brain Res Mol Brain Res.1999; 67 (1): 107-123) strengthen signal conduction in the cell, the IMP-fusion rotein that therefore exists with the homodimer form has more potentiality, in conjunction with more insulin receptor.
The IMP/IgG-Fc fusion rotein has hypoglycemic effect in the db/db mouse that is advanced by intramuscular injection, to obtain proof in vivo.This method has the advantage that in the time that continues several weeks, discharges continuously in the fusion rotein entering blood circulation.In the intramuscular injection IMP/IgG-Fc albumen db/db mouse, two weeks can detect the IMP fusion rotein in blood circulation later, but but detected the fusion rotein less than IMP in the proteic mouse of IgG-Fc as only injecting of contrast.
It is because it exists defective (Herberg and Coleman, Metabolism.1977 aspect the leptin signal transduction path as severe type ii diabetes model that Db/db is tried mouse; 26 (1): 59-99; People such as Chen, Cell.1996; 84 (3): 491-495).The glucose level that fusion rotein activation insulin receptor causes realizes normal.Normalizing that might blood sugar is to receive the activation of fusion rotein to insulin receptor more, and then has increased the metaboilic level of blood sugar in the body.In addition, although we do not see something of in the example effect (people such as Turton, the Nature.1996 that rodent model is treated observed reduction body weight with natural IMP; 379 (6560): 69-72).We do not observe IMP/IgG-Fc yet has the situation of apocleisis that (people such as Kim, Diabetes.2003 take place to animal; 52 (3): although 751-759) long-time and processing effectively insulin receptor agonist IMP has changed fasting blood glucose level (Wang and Brubaker, the Diabetologia.2002 of obesity mouse; 45 (9): 1263-1273).But body weight and peripheral insulin sensitivity still do not change (Wang and Brubaker, Diabetologia.2002; 45 (9): 1263-1273).This discovery has further supported only to have insulin resistant to be not enough to cause the generation of type ii diabetes, has only type ii diabetes (people such as Weyer, J Clin Invest.1999 just can take place when the β cell dysfunction; 104 (6): 787-794; Weyer A, Diabetes.1999; 48 (11): apocleisis effect 2197-2203) depends on effect (people such as Schick, the Am J Physiol Regul Integr Comp Physiol.2003 to a plurality of zones of cns brain; 284 (6): R1427-R1435) the fusion rotein ability that penetrates hemato encephalic barrier also need further be probed into.
Electrotransfer has been proved to be effectively and the method for safety in intramuscular injection fusion rotein and the combination.This method is widely used, and be used for cytokine, peptide hormone, soluble receptors and many embrane-associated proteins or cytoplasm protein.Really so, this method is being injected such as particularly useful aspect this proteinoid class regulatory factor of IMP/IgG-Fc fusion rotein systemicly.The advantage of IgG integration technology is the IMP/IgG-Fc fusion rotein that can obtain the laboratory scale amount in testing laboratory through a simple step program.IMP bacterial expression detected result shows that the expression efficiency of bacterial expression system will be lower than the expression efficiency of mammalian cell expression system, and this possibly ascribe at the protein of expression in escherichia coli than the false folding (misfold) of CHO-S cell expressing.Though the correct folding and functionalization albumen that chi Rosetta gami 2 bacterial cells are expressed improves (people such as Pnnz, J Biol Chem.1997 greatly compared with other bacteriums systems; 272 (25): 15661-15667) this raising is through increasing the formation that albumen two sulphur are good in the bacterial cell plastid and providing the tRNAs of the special codon that lacks in the intestinal bacteria system to realize (Kurland and Gallant, Curr Opin Biotechnol.1996; 7 (5) 489-493).
In a word, to be IMP (the insulin receptor agonist that obtains through display technique of bacteriophage) with IgG-Fc and two mutants thereof merge forms IMP/IgG-Fc and exist with the form of natural divalence to increase the transformation period, strengthen active in vivo and to reduce immunogenicity IMP/IgG-Fc fusion rotein in the present invention.In one embodiment of the invention, the IMP/IgG-Fc fusion rotein can directly be used through injection.Show that with the in vivo tests of mouse diabetes model can use through the injection of fusion rotein, this method can effectively be controlled intravital glucose level, avoids the generation of insulin resistant; Can treat mellitus in type ii diabetes db/db mouse model proof.Simultaneously, the drug metabolism experiment through rhesus monkey confirms that further IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) have the transformation period in the longer body than IMP/IgG-Fc.
Above-mentioned disclosed content has been carried out description substantially to the present invention.Understand through the present invention being done more comprehensively with reference to following specific embodiment.These embodiment only are for further explanation, rather than in order to limit scope of the present invention.In other words, some pro forma variations or replacement are to consider as what express one's ideas.Therefore, routine hereinafter some special terms of quoting are intended to express one's ideas, and the purpose of unrestricted its meaning.
Embodiment
Embodiment 1: the structure of carrier
Use overlapping PCR (overlap PCR) to make up the carrier of the encoding fusion protein that contains IMP and IgG1-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) carrier.IgG4-Fc contains in the district the constant heavy chain of IgG4 (hinge-CH2-CH3 part).Mouse IgK secretion leading peptide sequence and IMP sequence are merged so that guiding synthetic fusion rotein is secreted in the nutrient solution.The cDNA of coding IMP/hIgG-Fc fusion rotein is chemosynthesis; And be connected on the product of pcr amplification of coding human IgG4-Fc (hinge, CH2 and CH3), insert then between EcoRV and the BamHI site of pcDNA3.1 carrier and make up the IMP/hIgG-Fc/pcDNA3.1 carrier.But excretory IMP/hIgG-Fc and mutant fusion protein thereof contain the constant heavy chain of IgG4 (hinge-CH2-CH3).Mouse IgK secretion leader peptide sequence and IMP sequence merge guiding synthetic PE in cell culture fluid.This method guarantees that in secretion process after secretion guiding peptide was excised, the aminoterminal of the IMP of fusion rotein can merge with an active histidine residues.Fig. 1 has shown the diagram of excretory IMP/IgG-Fc fusion rotein.Be characterized in:
1. solved short problem of IMP transformation period; Because fusion rotein is the form excretory with homodimer; Have the long transformation period in vivo and tire (people such as Ozmen, J Immunol.1993,150 (7): 2698-2705 owing to the high affinity of part has reached height; People such as Kurschner, J Immunol.1992; 149 (12): 4096-4100; Kurschner etc., J Biol Chem.1992; 267 (13): 9354-9360).
2. because the plain acceptors of most of pancreas islet are to be pre-formed dimer (people such as George, Nat Rev Drug Discov.2002 at cell surface; 1 (10): 808-820; People such as Dupuis, Brain Res Mol Brain Res.1999; 67 (1): 107-123) thereby polypeptide and its acceptor to combine will be more effective;
3. the purification process in helping producing can pass through A albumen polydextran gel single step purification (people such as Jungbauer, Chromatogr.1989; 476:257-268).
Use gene-specific primer and overlapping PCR from IMP (chemosynthesis, Shenggong, Shanghai, China) cDNA and IgG plasmid amplification total length IMP and IgG-Fc cDNA.For overlapping PCR of the first round, use 5 '-GATATCATGGTGAGCACCCCCGAGTTCCTGGTGTTCCTGCTGTTCTGGATCCCCGC CAGCCGGGGCAGCCTGGAGG-3 ' and 5 '-TGCTGACCAAGCTCGCCGTCGACCCGCCGCCGCCGTCGCCG-3 '; 5 '-AGCGGCAGCTGGGCGGCGGCGGCAGGGCGGCGGCGGCAG-3 ' and 5 '-TGATGTGGGTCTTCTCGGACTCGGACTCGGACCCGTTCCCTAAG-3 '.Take turns among the overlapping PCR second and to use PCR and product to prepare subsequent use IMP/IgG-Fc cDNA.The product subclone of amplification is arrived carrier B am HI and Eco RV site, use 5 '-ATGGTGAGCACCCCCGAGTTCCTGGTGTTCCTGCTGTTCTGGATCCCCGCCAGCCG GGGCAGCCTGGAGG-3 ' and 5 '-TGATGTGGGTCTTCTCGGACTCGGACTCGGACCCGTTCCCTAAG-3 ' primer.
For making up the control vector of coding IMP-IgG-Fc; Use 5 '-GGCGGCGGCGGCAGCGGCGGCGGCG GCAG-3 ' and 5 '-TGATGTGGGTCTTCTCGGACTCGGACTCGGACCCGTTC-3 ', through increase separately IgG cDNA and be cloned into carrier B am HI and Eco RV site of PCR.Be used for coding human IgG4-Fc (primer of the segmental pcr amplification of cDNA of hinge-CH2-CH3) is: 5 '-GGCGGCGGCGGCAGCGGCGGCGGCGGCAG-3 ' and 5 '-TGATGTGGGTCTTCTCGGACTCGGACTCGGACCCGTTCCCTAAG-3 '.Carrier contains CMV direct-early stage (immediate-early) enhanser and promotor, single eukaryote transcriptional units.Purine tail and transcription termination sequence.For making the secretion of IMP sequence, introduce mouse IgK-chain signal peptide sequence in its 5 ' end through PCR.Be in bacterium, to express the IMP/IgG-Fc fusion rotein, merge the cDNA sequence through pcr amplification and subclone to pET32a carrier (Novagen, EMD Bioscience, San Diego, CA).The construction process of IgG-Fc (QL) and IMP/IgG-Fc (QA) expression plasmid is the same; T250Q/M428L and T307Q/N434A mutational site be two pairs of PCR primers of design in addition; Carry out overlapping PCR reaction, obtain IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) expression plasmid (Fig. 1) at last.
Embodiment 2:IMP/IgG-Fc, the expressing cho cell of IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) fusion rotein
Expressing and the ability of secreting the IMP/IgG-Fc fusion rotein, transient expression in fusion rotein carrier transfection to the CHO-S cell of structure in order to evaluate carrier.48h after the transfection extracts total RNA and assesses expression effect with RT-PCR in transfectional cell.The applying gene Auele Specific Primer detects the expression of IMP/IgG-Fc antigen-4 fusion protein gene on transcriptional level and the expression of IgG-Fc crt gene (Fig. 2 A).In control sample, do not detect the expression of transcriptional level.
The CHO-S cell lysate and the cell culture fluid that have detected transfection with Western immunity marking method and anti-mouse antibody are with the definite expression of fusion rotein on translation skill.Shown in Fig. 2 B, in nutrient solution and cell lysate, detect the Fc fusion rotein simultaneously.Fusion rotein greatly about 35kDa, is the monomeric molecular weight of fusion rotein under reductive SDS-PAGE condition in the electrophoretic migration position.In conditioned medium and cell lysate, detect fusion rotein simultaneously and show that fusion rotein is at the synthetic justacrine of Mammals detail.
Expression in mammalian cell is that (CA) transfection is in the CHO-S cell for Invitrogen, Carlsbad through liposome (Lipofectamine) 2000 with the cDNA of IMP/IgG-Fc or IgG-Fc.Its process is to be every hole 2.5 * 10 with density earlier 5Individual CHO-S cell is grown in 6 porocyte culture plates.The DMEM (Invitrogen) that adds the serum-free contain 4 μ gIMP/IgG-Fc cDNA and 10 μ l transfection liposomes and do not have microbiotic cultivates.Behind the transfection 6h, convert full nutrient solution into.48h collects nutrient solution and cell respectively after the transfection.In order to express the IMP/IgG-Fc fusion rotein on a large scale, be grown in CHO-S cell in the 150mm petridish with the positively charged ion transfection reagent, gather acetimide) (Poly (ethyleneimine) (and PEI, 25kDa) relevant with 80 μ g.The cDNA transfection.With 150Mm Nacl difference dilution DNA and PEI, mix the back and cultivated 20 minutes.Then the DNA/PEI mixture is added in the cell, and in serum-free does not have the nutrient solution of microbiotic, cultivates 6h.To be added with the DMEM replacement nutrient solution of 10%FBS and P/S.The transfection efficiency of this method can be up to 85%.
For setting up the CHO-S cell of stably express IMP/IgG-Fc, will be at 6 porocyte culture plates (2.5 * 10 5Individual cells/well) cell of going up growth is with linearizing IMP/IgG-Fc of 4 μ g or IgG-Fc transfection.Behind the transfection 24h, cell arrives the cell in its genome with the recombinant plasmid stable integration containing division of DMEM nutrient solution and the cultivation of G418 (500 μ g/ml) to screen those.In the culturing process, changed nutrient solution and once form in per 3 days until cell clone.Separate monoclonal cell and be extended to stable clone and the supernatant of tissue culture that these clones are grown detects fusion rotein with SDS-PAGE or Elisa on 24 orifice plates.Selection can be secreted fusion rotein in a large number, and the stable cell that goes down to posterity is as later specificity analysis.
Embodiment 3: purifying IMP/IgG-Fc from the mammalian cell nutrient solution, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) fusion rotein
For carrying out small-scale purifying, the nutrient solution that will collect from transfectional cell (be generally 6 well culture plates every hole get 2.5ml) add with the damping fluid that contains 100mM Tris pH value 8.0 and 150mM Nacl in advance 70 μ l of flushing expire volume (packed volume).The quick wash-out resin of the proteic polydextran gel of A (Amersham-Pharmacia, Piscataway, NJ) in.After 4 ℃ of following overnight cultures, with Tris damping fluid flushing, the sample buffer that contains SDS with 30 μ l then is directly with albumen wash-out from the resin.
For obtaining a large amount of fusion roteins, use the protein purification that A albumen sephadex column can carry out middle scale.The transfection IMP/lgG-Fc that the column volume of 50ml is grown in can purifying 15cm petridish, the conditioned medium of the CHO-S cell of IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) fusion vector.The 50ml DMEM nutrient solution of 48h after the collecting cell transfection, or the cell of collection stably express fusion rotein add the full volume of A albumen polydextran gel 1mL with it, (Amersham-Pharmacia).Add 1%TritonX-100 4 ℃ of following overnight cultures.With the PBS damping fluid washing that contains 1%TritonX-100, wash with 150mM Nacl then then, use 1mM Nacl Padil (pH2.7) at last albumen wash-out from the resin.Elutriant uses Tris damping fluid (pH9.0) neutralization and purified proteins also to use the PBS wash-out with PD-10 gel column desalination (Amersham-Pharmacia) immediately.Like finding (Fig. 2 A) among the figure, two step elution processs can be with most of fusion rotein by eluting on the sephadex column.The component of sample is developed through the SDS-PAGE dissolving and through Coomassie brilliant blue dyeing, and (result shows every ml nutrient solution inoculation 1.25 * 10 to assess productive rate and purifying rate 5Behind the cell cultures 2d, the fusion rotein yield is about 6 μ g/ml).
Embodiment 4:IMP/IgG-Fc, IMP/IgG-Fc (QL) and the proteic bacterial expression of IMP/IgG-Fc (QA)
In Bacillus coli cells, express IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) fusion rotein.For the codon bias (Codon bias) that compensates the e. coli bl21 cell; Used and to have promoted disulfide linkage to form and can the Rosetta gami 2 cell (Mercks of extra lift-launch plasmid to express 7 rare tRNAs; Germany) use IMP/IgG-Fc/pET32a; IMP/IgG-Fc (QL) pET32a behind IMP/IgG-Fc (QA) pET32a or IgG-Fc/pET32a carrier (Novagen) transform bacteria, selects and filters out wherein several clones with the optimum expression fusion rotein.Protein expression is that monospecific polyclonal is cultivated in 2 * YT of the 50ml that kantlex is arranged nutrient solution in 37 ℃ of incubated overnight.Then nutrient solution is diluted to (1: 50) new nutrient solution, reaches OD until cell density 600Behind the reading value 0.6, the IPTG (EMD) 3 hours that in nutrient solution, adds 1mM again is with abduction delivering.Collect bacterium and bacterium ball (Pallet) is preserved to carry out other steps.
Proteinic extraction is the bacterium after centrifugal to be suspended in the proteinase inhibitor cocktail that contains of ice bath again (Sigrma, St.Louis use the ultrasonic treatment cell after PBS damping fluid MO) in brief.Add 1%TritonX-100 with soluble protein, centrifugal behind the 30min (12000g 10min) collects the supernatant that contains fusion rotein.The fusion rotein of expressing is through A albumen polydextran gel purifying and pass through SDS-PAGE; With Coomassie brilliant blue staining analysis checking (data not shown).From the inoculum of 3L, can purify IMP/IgG-Fc and the Fc-only fusion rotein of about 120 μ g.In mammalian cell of expressing the IMP fusion rotein and bacterium, all can observe the IMP level and be the increase of peptide dose-dependently.But find that the expression level of total IMP in bacterium is lower than the level in mammalian cell.
Embodiment 5:IMP/IgG-Fc, the generation of (type ii diabetes model) mellitus in IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) the treatment prevention db/db mouse
The female db/db mouse in 4 ages in week (Weitong lihua, Beijing, China).The contrast mouse CJ57BLKS of age background coupling and CD1 (Weitong lihua, Beijing, China).Being tried mouse is having raising under control illumination (12 illumination/12 hour dark) and the constant temperature, and competent (suitable rodents) food and drinking-water is provided.The rules that all programs all according to Chinese experimental animal Ethics Committee is carried out and is ratified through laboratory animal Ethics Committee.
Handle mellitus db/db through foregoing intramuscular injection and tried mouse (Prud ' homme and Chang, Gene Ther.1999; 6 (5): 771-777).In brief, having been tried the intramuscular injection of mouse tibiofibula and contained the PBS of the IMP/IgG-Fc fusion rotein of 1nM anaesthetic treatment, injection uses No. 27 syringe needles of plastic jacket to be 5mm to limit the needle injection degree of depth.Monitor its body weight and fasting blood glucose level every day.Got the saphena blood under the condition on an empty stomach behind the protein injection in 2,3,5,7 days.Basic blood-glucose meter with Roche company produces is measured fasting blood glucose level, IMP, and IMP/IgG-Fc, the measurement of IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) level is described below:
The intravital expression amount of IMP/IgG-Fc fusion rotein is to detect the active IMP content of blood plasma with the IMP Elisa test kit that this research institution makes and encapsulates to assess.As shown in the figure, for the first time inject 2 days after, injection IMP/IgG-Fc tried mouse inject IgG-Fc to be tried mouse serum I MP level significantly high.The IMP level of IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) fusion rotein and IMP/IgG-Fc basically identical (Fig. 5).
In process of the test, the significant (not shown) that changes does not take place in two groups of body weight of being tried mouse.In first month after the injection, two groups are tried the mouse fasting blood glucose level and are not also had significant difference (not shown).Yet in 1 week after injection, the mouse fasting blood glucose level that tried of injecting IMP/IgG-Fc significantly is lower than control group (P＜0.05) (Fig. 5).This result shows, the IMP/IgG-Fc of injection in the body, and IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) are tried the effect that mouse has lowering blood glucose to db/db, and its reason is likely owing to activated insulin receptor, promotes the metabolism of intravital carbohydrate.
Embodiment 6:IMP/IgG-Fc, the generation of mellitus in IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) the prevention STZ inductive insulin deficit property mouse type i diabetes model
Nearest result of study shows, insulin-simulated peptide (IMP) can simulate the Regular Insulin blood sugar regulation alleviate type i diabetes (people such as Maja Jensen, J Biol Chem.2007,282:35179-35186).In order to confirm the validity of IMP/IgG-Fc fusion rotein to the pancreatic damage model, we have studied its effect to U-9889 (STZ) inductive mellitus CD1 mouse.Fusion rotein (1nM/ mouse) intramuscular injection of IMP/IgG-Fc, IgG-Fc or IgG-Fc of will encoding is respectively tried mouse to CD1.The injection fusion rotein simultaneously; Continuous 5 days injection STZ (55mg/kg; I.p.) result shows; IgG-Fc control group blood sugar significantly increases, and reaches the level (blood sugar >=17mM), but the mouse of being tried of injecting IMP/IgG-Fc or IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) fusion rotein is protected the low incidence (Fig. 4) that shows obvious mellitus of mellitus in several days.
Embodiment 7:RT-PCRization method detects IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) Expression of Fusion Protein
(Qiagen, Valencia CA) detect the expression of IgG fusion rotein on transcriptional level with gene-specific primer to use single stage method RT-PCR test kit.For detecting IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) antigen-4 fusion protein gene are expressed, and extract the Auele Specific Primer of cell total rna 100ng as template and interpolation 0.6 μ M:
(SEQ IDNo：18)
5′-AGCCTGGAGGAGGAGTGGGCCCAGATCGAGTGCGAGGTGTACGGCCGGGGCTGC-3′
With
(SEQ ID No：19)
5′-TCGAAGATGATGACCAAGATCGCCGTCGAC-3′
For detecting the expression of IgG Fc, use Auele Specific Primer simultaneously
(SEQ ID No：20)
5 '-GAGAGCAAGTACGGCCCCCCCTGCCCCAGCTGCCCCGCCCCCGAGTTCCTGGGCGG CCCC-3 ' with
(SEQ ID No：21)
5′-TCGACGTCGCACTACGTGCTCCGGGACGTGTTGGTGATGTGGGTCTTCTCGGACTCGGACTCGGACCCGTTC-3′)
The condition of one one step RT-PCR is 50 ℃ of 30min, 95 ℃ of 5min, and 94 ℃ of 30min of 40 round-robin, 55 ℃ 30 seconds and 72 ℃ 60 seconds carry out the extension of 10min subsequently under 72 ℃.The RT-PCR product separates through 1% agarose gel electrophoresis, the Goldenview detection of dyeing.
Embodiment 8:SDS-PAGE and/or immunoblotting detect IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) fusion rotein
Fusion rotein to expressing carries out the detection on the translation skill.Get the fusion rotein that the is dissolved in the SDS sample-loading buffer 30 μ l that extract on a small scale and after 10%SDS PAGE gel electrophoresis separates, be transferred to CAM; And with anti-mouse antibody (1: 5000; Amersham-Pharmacia) survey, then separate the back with Coomassie brilliant blue dyeing detection through 10%SDS PAGE gel electrophoresis with the develop fusion rotein of the medium-scale extraction of (Amersham-Pharmacia) 30 μ l of ECL.
Experiment 9: pharmacokinetics detects
Especially the transformation period is detected to medicine kinetics in vivo through the experiment of rhesus monkey pharmacokinetics.Choose 9 of healthy adult rhesus monkeies, male and female half and half, subcutaneous injection IMP, IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA), the concentration of blood Chinese traditional medicine is detected in the back with the ELISA method.Concentration-time data calculates through pharmacokinetic parameter, shows that the medicine elimination meets two chamber models, and Plasma Concentration-time curve and each medicine are seen table 1 for parameter.Wherein, eliminating the transformation period in the IMP/IgG-Fc injection groups, the average body of 9 rhesus monkeies is 98 ± 19.2h.The average out to peak concentration is 24.0 ± 4.2 μ g/ml, and on average eliminating speed is 0.190 ± 0.026ml/kg/h, and area under the drug-time curve is 4,915 ± 539 μ g.h/ml, and the transformation period significantly is longer than 7 ± 1.8h of IMP in the body.While IMP/IgG-Fc (QL) is prolong half-life to 153 ± 41.5h further.
Table 1:The pharmacokinetic parameter of IMP/IgG-Fc
Figure BSA00000629344100201
Statistical analysis:All data are all represented with the form of mean+SD (mean+SEM).(Statistical Analysis Systems, Cary NC) carry out student t check or variance analysis (ANOVA), with " n-1 " test of hypothesis (post hoc custom hypotheses test), with p＜0.05 expression significant difference with SAS software.
Sequence table:
IMP, human IgG1-Fc, human IgG2-Fc, human IgG 3-Fc, human IgG 4-Fc and two mutants (QL) thereof and (QA), mouse IgK homing sequence:
< 110>Haitao Zhang
< 120>insulin-simulated peptide fusion protein and two mutants and application thereof
<160>
<210>1
<211>30
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<310>
<311>2011-11-26
<312>
<400>1
Ser Leu Glu Glu Glu Trp Ala Gln Ile Glu Cys Glu Val Tyr Gly
1 5 10 15
Arg Gly Cys Pro Ser Glu Ser Phe Tyr Asp Trp Phe Glu Arg Gln
16 20 25 30
<210>2
<211>5
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<400>2
Gly Gly Gly Gly Ser (SEQ ID NO：2)
<210>3
<211>10
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<400>3
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID NO：3)
<210>4
<211>15
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<400>4
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID NO：4)
<210>5
<211>20
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<400>5
(GlyGlyGlyGlySer)4 (SEQ ID NO：5)
<210>6
<211>25
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<400>6
(GlyGlyGlyGlySer)5 (SEQ ID NO：6)
<210>7
<211>30
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<400>7
(GlyGlyGlyGlySer)6 (SEQ ID NO：7)
<210>8
<211>4
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<400>8
(GlyGlyGlySer) n wherein n be 0,1,2,3,45 or 6.(SEQ ID NO：8)
<210>9
<211>237
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<400>9
IgG1-Fc(Hinge-CH2-CH3)：(SEQ ID NO：9)
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
16 20 25 30
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
31 35 40 45
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
46 50 55 60
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
61 65 70 75
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
76 80 85 90
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
91 95 100 105
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
106 110 115 120
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
121 125 130 135
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
136 140 145 150
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
151 160 165 170
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
171 175 180 185
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
186 190 195 200
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
201 205 210 215
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
216 220 225 230
Leu Ser Leu Ser Pro Gly Lys
231 235
<210>10
<211>228
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<400>10
IgG2-Fc(Hinge-CH2-CH3)：(SEQ ID NO：10)
Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro
1 5 10 15
Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
16 20 25 30
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
31 35 40 45
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
46 50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
61 65 70 75
Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val
76 80 85 90
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
91 95 100 105
Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
106 110 115 120
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
121 125 130 135
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
136 140 145 150
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
151 155 160 165
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp
166 170 175 180
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
181 185 190 195
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
196 200 205 210
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
211 215 220 225
Pro Gly Lys
226
<210>11
<211>249
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<400>11
IgG3-Fc(Hinge-CH2-CH3)：(SEQ ID NO：11)
Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg
1 5 10 15
Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg
16 20 25 30
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
31 35 40 45
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
46 50 55 60
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
61 65 70 75
Gln Phe Lys Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
76 80 85 90
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Phe Arg Val Val
91 95 100 105
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
106 110 115 120
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
121 125 130 135
Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val
136 140 145 150
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
151 155 160 165
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
166 170 175 180
Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr
181 185 190 195
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
196 200 205 210
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe
211 215 220 225
Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln
226 230 235 240
Lys Ser Leu Ser Leu Ser Pro Gly Lys
241 245
<210>12
<211>229
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<400>12
IgG4-Fc(Hinge-CH2-CH3)：(SEQ ID NO：12)
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu
1 5 10 15
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
16 20 25 30
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
31 35 40 45
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
46 50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
61 65 70 75
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Arg Val Leu Thr Val
76 80 85 90
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
91 95 100 105
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
106 110 115 120
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
121 125 130 135
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
136 140 145 150
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
151 155 160 165
Asn Gly Gln Pro Glu Asp Asn Tyr Lys Thr Thr Pro Pro Val Leu
166 170 175 180
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
181 185 190 195
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
196 200 205 210
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
211 215 220 225
Ser Leu Gly Lys
226
<210>13
<211>247
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<400>13
Sudden change IgG1-Fc (Hinge-CH2-CH3): (SEQ ID NO:13)
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
16 20 25 30
Lys Pro Lys Asp Xaa1 Leu Met Ile Ser Arg Thr Pro Glu Val Thr
31 35 40 45
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
46 50 55 60
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
61 65 70 75
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
76 80 85 90
Leu Xaa2 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
91 95 100 105
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
106 110 115 120
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
121 125 130 140
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
141 145 150 155
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
156 160 165 170
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
171 175 180 185
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
190 195 200 205
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
210 215 220 225
Ser Val Xaa3His Glu Ala Leu His Xaa4 His Tyr Thr Gln Lys Ser
226 230 235 240
Leu Ser Leu Ser Pro Gly Lys
241 245
<210>14
<211>227
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<400>14
Sudden change IgG2-Fc (Hinge-CH2-CH3): (SEQ ID NO:14)
Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro
1 5 10 15
Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
16 20 25 30
Xaa1 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
31 35 40 45
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
46 50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
61 65 70 75
Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Xaa2 Val Val
76 80 85 90
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
91 95 100 105
Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
106 110 115 120
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
121 125 130 135
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
136 140 145 150
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
151 155 160 165
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp
166 170 175 180
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
181 185 190 195
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Xaa3 His
196 200 205 210
Glu Ala Leu His Xaa4 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
211 215 220 225
Pro Gly Lys
226
<210>15
<211>249
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<400>15
Sudden change IgG3-Fc (Hinge-CH2-CH3): (SEQ ID NO:15)
Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg
1 5 10 15
Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg
16 20 25 30
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
31 35 40 45
Pro Pro Lys Pro Lys Asp Xaa1 Leu Met Ile Ser Arg Thr Pro Glu
46 50 55 60
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
61 65 70 75
Gln Phe Lys Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
76 80 85 90
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Phe Arg Val Val
91 95 100 105
Ser Val Leu Xaa2 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
106 110 115 120
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
121 125 130 135
Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val
136 140 145 150
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
151 155 160 165
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
166 170 175 180
Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr
181 185 190 195
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
196 200 205 210
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe
211 215 220 225
Ser Cys Ser Val Xaa3 His Glu Ala Leu His Xaa4 Arg Phe Thr Gln
226 230 235 240
Lys Ser Leu Ser Leu Ser Pro Gly Lys
241 245
<210>16
<211>234
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<400>16
Sudden change IgG4-Fc (Hinge-CH2-CH3): (SEQ ID NO:16)
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu
1 5 10 15
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
16 20 25 30
Asp Xaa1 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
31 35 40 45
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
46 50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
61 65 70 75
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Arg Val Leu Xaa2 Val
76 80 85 90
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
91 95 100 105
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
106 110 115 120
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
125 130 135 140
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
141 145 150 155
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
156 160 165 170
Asn Gly Gln Pro Glu Asp Asn Tyr Lys Thr Thr Pro Pro Val Leu
171 175 180 185
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
186 190 195 200
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Xaa3
201 205 210 215
His Glu Ala Leu His Xaa4 His Tyr Thr Gln Lys Ser Leu Ser Leu
216 220 225 230
Ser Leu Gly Lys
231
<210>16
<211>234
<212>PRT
< 213>artificial sequence
<220>
< 223>synthetic construct
<400>16
Mouse IgK signal leader：(SEQ ID NO：17)
Met Val Ser Thr Pro Glu Phe Leu Val Phe Leu Leu Phe Trp Ile
1 5 10 15
Pro Ala SerArg Gly
16 20

Claims (10)

1. fusion rotein; Formed through connection peptides (Linker) and IgG-Fc or IgG-Fc two mutants by insulin-simulated peptide, its general formula is IMP (Insulin mimetic peptides)-Linker-IgG-Fc or IgG-Fc-Linker-IMP (Insulin mimetic peptides).
2. the said IMP of claim 1 is insulin-simulated peptide and two mutants (SEQ ID NO:1) thereof.
3. the said Linker of claim 1 is selected from sequence SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; Or SEQ ID NO:7, (GGGGS) n, wherein n is 1,2,3,4,5 or 6.
4. the said Linker of claim 1 can be selected from SEQ ID NO:8, and wherein n is 1,2,3,4, and 5 or 6, perhaps directly connect, perhaps be the connection peptides of other glycocoll enrichment.
5. (hinge area-CH2-CH3) sequence is Fc and sudden change or the deletant of human IgG1, IgG2, IgG3 or IgG4 to the said IgG Fc of claim 1, and above-mentioned sequence is selected from SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11 or SEQ ID NO:12 perhaps are selected from the heterozygote of above-mentioned IgG-Fc sequence, and wherein the IgG-Fc sequence is numbered with reference to EU (Kabat) index, and importantly above-mentioned IgG-Fc two mutants is selected from sequence SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:16, the said mutation site can be unit point sudden change or multisite mutation combination, wherein:
Xaa 1=Gln or Thr
Xaa 2=Gln or Thr
Xaa 3=Leu, Met or His
Xaa 4=Ala or Asn
6. according to claim 1, further, the fusion rotein that links to each other with insulin-simulated peptide (IMP) can be selected from rHSA or Transferrins,iron complexes, also can be selected from rHSA or Transferrins,iron complexes part fragment, two mutants and deletant.
7. the said fusion rotein of claim 1～5 is applied to the prevention and the treatment of people I type and type ii diabetes, and perhaps its dna sequence dna is applied to gene therapy.
8. the said fusion rotein two mutants of claim 5 is based on to increase with the avidity of FcRn and comes further to increase the IMP transformation period in vivo.
9. the said fusion rotein of claim 1～5 uses eukaryotic cell to express, and uses A or G albumen sephadex chromatography post in the protein purification process.
10. the said fusion rotein of claim 1～5 uses prokaryotic cell prokaryocyte to express.
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CN110172103B (en) * 2016-09-29 2023-05-09 上海凯茂生物医药有限公司 GLP-1 analogue-Fc fusion protein, and preparation method and application thereof
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